Frequently, advanced prostate tumors become androgen-independent. The goal of this project is to determine the sequence of events which lead to the autonomous state. Our hypothesis is that these tumors may have escaped androgen control not because of escape from regulation by androgen receptor (AR) per se, but rather because the receptor has become activated by one of several novel pathways. AIM 1: An AR mutation could change its steroid specificity so that it becomes stimulated by different ligands. Mutations, mRNA splicing changes or deletions could cause expression of active, truncated AR forms. We propose to determine whether somatic mutations in tumor androgen receptors exist which account for the androgen-independent state. Mutations will be sought first simply by Western immunoblots of tumor extracts to see if truncated receptor is present. We will evaluate the androgen receptor gene from these samples by Polymerase Chain Reaction (PCR) to detect either altered RNA processing or gene mutations. If mutants are found, these receptors will be cloned for detailed experimental analysis in cultured cells. AIM 2: Some receptors can be activated in tissue culture cells by other effector substances which modulate intracellular protein phosphorylation pathways. This mechanism, termed "ligand-independent receptor activation", may also occur for androgen receptor. If so, effector substances such as growth factors or peptide hormones could stimulate AR. We propose to test whether androgen receptors are subject to ligand-independent activation in hormone-independent prostate tumors. We will determine whether we can modulate receptor activity by protein kinase/phosphatase alterations. If this pathway is seen, we will try to define which trophic substances are affecting receptor phosphorylation. AIM 3: The steroid receptor superfamily proteins frequently are gene regulatory molecules. Some of them are well-known. Others have been obtained by screening cDNA libraries, and are termed "orphan receptors". No systematic study has been done to detect and identify these factors in prostate tumors. We propose to isolate and identify new orphan members from human prostate mRNA, cDNA libraries and/or from genomic libraries. We will probe the libraries using sets of degenerate oligonucleotides as primers to direct PCR amplification of desired sequences. The initial screenings will use mRNA pools as well as cDNA libraries, since they by definition represent mRNAs of expressed genes in the prostate tissue. Coincident with this approach will be attempts to enrich the libraries for orphans expressed late in the tumor progression, by subtractive hybridization. This approach may also permit us to isolate genes specific for prostate cancer progression. We propose to assay for expression of these new "orphans" in other prostate tumor samples, to see if their expression correlates with the androgen-autonomous state. AIM 4: The orphan receptor gene targets will not be known. We propose to test a remarkably simple amino acid alignment strategy, using the known amino acid contacts made to the DNA by the glucocorticoid receptor to define the important amino acids in the orphans. In preliminary studies, we observed that a single amino acid defines the choice of DNA response element to which known receptors bind. We will test that system using the orphans identified in this project, using orphan-directed induction of reporter genes in DU145 (AR-) and LnCAP (AR+) cells. If initial studies support our hypothesis, we will then perform site-directed mutagenesis to confirm the functional significance of the amino acid predictions. In summary, we have defined several inter-related aspects of cell control by this receptor family, as they apply to the prostate cancer problem. We believe that our expertise in the area of steroid receptor studies makes this project particularly exciting and challenging.